LOCUS       sequence-295619-        8099 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression plasmid for SARS-CoV-2 nsp4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8099)
  TITLE     SARS-CoV-2 protein Mammalian expression constructs, tagged, for 
            protein expression and/or purification
REFERENCE   2  (bases 1 to 8099)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pGBW-m4133987
FEATURES             Location/Qualifiers
     source          1..8099
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1508..1528
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1529..1570
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     polyA_signal    1639..1760
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1650..1669)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1704..1723
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      1875..1921
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      1951..2087
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    2132..2203
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(2429..3017)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2509..2528)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3517..4726
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     4563..4582
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     CDS             4730..5758
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     polyA_signal    5785..5833
                     /note="synthetic polyadenylation signal"
     CDS             join(6099..6167,6168..6959)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6317..6336)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      6972..7108
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    7253..7324
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(7329..7375)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    7393..7441
                     /note="synthetic polyadenylation signal"
     enhancer        7522..7825
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7827..8029
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7980..8000
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 caccatgaag attgtaaata actggttgaa acaactaata aaggttactt tagtgttcct
       61 gtttgtcgcc gcaatcttct atcttatcac cccggtgcac gtgatgagta aacatacaga
      121 cttttcttca gaaattatcg ggtacaaggc tatcgatggc ggtgtaacgc gcgacattgc
      181 ctccacagat acctgcttcg cgaacaaaca cgctgacttt gatacttggt tcagccagcg
      241 aggaggctcg tacaccaatg acaaagcctg tccactgatc gcagccgtga taactagaga
      301 ggttggattc gtcgtccctg ggctccccgg gacaattctc cgtacaacca acggcgactt
      361 tctgcatttt ctgccccggg tgttctccgc agtcggtaat atctgttata ctccatctaa
      421 gctcattgag tacacggatt ttgctaccag cgcgtgcgtg ttggccgcag aatgcaccat
      481 atttaaggac gcttctggaa aacctgtgcc ctattgttac gataccaatg tgcttgaggg
      541 ctcagttgcc tatgaaagtc tgaggccaga cactagatac gttttaatgg atggaagcat
      601 catccagttc ccaaacacgt atctggaggg gtccgtgcgg gtcgtgacca cattcgattc
      661 agaatactgc cgccacggta catgcgagag gtctgaggct ggcgtatgtg tgtcaactag
      721 cggtcgatgg gttctaaata acgattatta tcgttctctg cccggcgttt tttgtggagt
      781 ggacgccgtt aaccttttga ccaacatgtt tacacctctc attcagccca ttggtgcact
      841 ggatatctcc gcgagtattg tggctggggg aatagtcgcc attgtcgtaa cctgcttggc
      901 atactatttc atgcgtttca ggcgagcttt tggcgaatac tcacatgtcg tggccttcaa
      961 tactcttctg tttctgatga gcttcacggt actctgtctg accccagtat acagtttcct
     1021 acctggggtg tattctgtga tctacttata cctcacattt tacctgacca atgacgtgtc
     1081 ctttctggcc cacatccaat ggatggttat gttcacgccg ctcgtgcctt tctggattac
     1141 aatcgcttat ataatctgta ttagcacgaa gcatttctat tggttctttt ccaactactt
     1201 aaagcggcgc gtcgtattta atggggtatc gtttagcact tttgaggaag ccgcgctctg
     1261 cactttcttg cttaataagg agatgtattt gaaattaaga tctgacgttc tgcttccttt
     1321 aacacagtac aacagatacc tggcgttgta taacaaatac aaatatttct cgggagccat
     1381 ggatacaact tcctatagag aggctgcttg ttgccacctt gcaaaggcac tcaacgactt
     1441 cagtaacagc ggcagtgatg tgctttatca gccccctcag acttcaataa catccgccgt
     1501 cctccaagaa aacctttact ttcagtcggg gaaacctata cccaatccac ttctcggctt
     1561 agattctacc taaggatccg caggcctctg ctagcttgac tgactgagat acagcgtacc
     1621 ttcagctcac agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     1681 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     1741 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     1801 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt attggcccat
     1861 ctctatcggt atcgtagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     1921 gtgcccctcg ggccggattg ctatctaccg gcattggcgc agaaaaaaat gcctgatgcg
     1981 acgctgcgcg tcttatactc ccacatatgc cagattcagc aacggatacg gcttccccaa
     2041 cttgcccact tccatacgtg tcctccttac cagaaattta tccttaaggt cgtcagctat
     2101 cctgcaggcg atctctcgat ttcgatcaag acattccttt aatggtcttt tctggacacc
     2161 actaggggtc agaagtagtt catcaaactt tcttccctcc ctaatctcat tggttacctt
     2221 gggctatcga aacttaatta accagtcaag tcagctactt ggcgagatcg acttgtctgg
     2281 gtttcgacta cgctcagaat tgcgtcagtc aagttcgatc tggtccttgc tattgcaccc
     2341 gttctccgat tacgagtttc atttaaatca tgtgagcaaa aggccagcaa aaggccagga
     2401 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     2461 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     2521 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     2581 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     2641 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     2701 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     2761 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     2821 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     2881 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2941 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     3001 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     3061 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     3121 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     3181 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     3241 catccatagt tgcatttaaa tttccgaact ctccaaggcc ctcgtcggaa aatcttcaaa
     3301 cctttcgtcc gatccatctt gcaggctacc tctcgaacga actatcgcaa gtctcttggc
     3361 cggccttgcg ccttggctat tgcttggcag cgcctatcgc caggtattac tccaatcccg
     3421 aatatccgag atcgggatca cccgagagaa gttcaaccta catcctcaat cccgatctat
     3481 ccgagatccg aggaatatcg aaatcggggc gcgcctggcc tccgcgccgg gttttggcgc
     3541 ctcccgcggg cgcccccctc gtcacggcga gcgctgccac gtcagacgaa gggcgcagga
     3601 gcgtcctgat ccttccgccc ggacgctcag gacagcggcc cgctgctcat aagactcggc
     3661 cttagaaccc cagtatcagc agaaggacat tttaggacgg gacttgggtg actctagggc
     3721 actggttttc tttccagaga gcggaacagg cgaggaaaag tagtcccttc tcggcgattc
     3781 tgcggaggga tctccgtggg gcggtgaacg ccgatgatta tataaggacg cgccgggtgt
     3841 ggcacagcta gttccgtcgc agccgggatt tgggtcgcgg ttcttgtttg tggatcgctg
     3901 tgatcgtcac ttggtgagta gcgggctgct gggctggccg gggctttcgt ggccgccggg
     3961 ccgctcggtg ggacggaagc gtgtggagag accgccaagg gctgtagtct gggtccgcga
     4021 gcaaggttgc cctgaactgg gggttggggg gagcgcagca aaatggcggc tgttcccgag
     4081 tcttgaatgg aagacgcttg tgaggcgggc tgtgaggtcg ttgaaacaag gtggggggca
     4141 tggtgggcgg caagaaccca aggtcttgag cccttcgcta atgcgggaaa gctcttattc
     4201 gggtgagatg ggctgggcac catctgggga ccctgacgtg aagtttgtca ctgactggag
     4261 aactcggttt gtcgtctgtt gcgggggcgg cagttatggc ggtgccgttg ggcagtgcac
     4321 ccgtaccttt gggagcgcgc gccctcgtcg tgtcgtgacg tcacccgttc tgttggctta
     4381 taatgcaggg tggggccacc tgccggtagg tgtgcggtag gcttttctcc gtcgcaggac
     4441 gcagggttcg ggcctagggt aggctctcct gaatcgacag gcgccggacc tctggtgagg
     4501 ggagggataa gtgaggcgtc agtttctttg gtcggtttta tgtacctatc ttcttaagta
     4561 gctgaagctc cggttttgaa ctatgcgctc ggggttggcg agtgtgtttt gtgaagtttt
     4621 ttaggcacct tttgaaatgt aatcatttgg gtcaatatgt aattttcagt gttagacttg
     4681 taaattgtcc gctaaattct ggccgttttt ggcttttttg ttagacaaca tgggtaaaaa
     4741 gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc
     4801 cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg
     4861 gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt
     4921 ttatcggcac tttgcatccg ccgcgctccc gattccggaa gtgcttgaca ttggggagtt
     4981 cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct
     5041 gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggcaatgg atgccatcgc
     5101 tgccgccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca
     5161 atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca
     5221 aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct
     5281 ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa
     5341 tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg
     5401 ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga
     5461 gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc cccggctccg
     5521 ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt
     5581 cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac
     5641 tgtcgggcgt acacaaatcg cccgcagaag cgccgccgtc tggaccgatg gctgtgtaga
     5701 agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaataagc
     5761 tagtatgtaa gcctagtctt agataataaa atcgctatcc atcgaagatg gatgtgtgtt
     5821 ggttttttgt gtgtgtaacg ctaggcgcgc ctggtgtacc gagaacgatc ctctcagtgc
     5881 gagtctcgac gatccatatc gttgcttggc agtcagccag tcggaatcca gcttgggacc
     5941 caggaagtcc aatcgtcaga tattgtactc aagcctggtc acggcagcgt accgatctgt
     6001 ttaaacctag atattgatag tctgatcggt caacgtataa tcgagtccta gcttttgcaa
     6061 acatctatca agagacagga tcagcaggag gctttcgcat gagtattcaa catttccgtg
     6121 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     6181 tggtgaaagt aaaagatgct gaagatcagt tgggtgcgcg agtgggttac atcgaactgg
     6241 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgcttt ccaatgatga
     6301 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     6361 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtattca ccagtcacag
     6421 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6481 gtgataacac tgcggccaac ttacttctga caacgattgg aggaccgaag gagctaaccg
     6541 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6601 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacct
     6661 tgcgtaaact attaactggc gaactactta ctctagcttc ccggcaacag ttgatagact
     6721 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6781 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     6841 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6901 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6961 cgattctagg tgcattggcg cagaaaaaaa tgcctgatgc gacgctgcgc gtcttatact
     7021 cccacatatg ccagattcag caacggatac ggcttcccca acttgcccac ttccatacgt
     7081 gtcctcctta ccagaaattt atccttaaga tcgtttaaac tcgactctgg ctctatcgaa
     7141 tctccgtcgt ttcgagctta cgcgaacagc cgtggcgctc atttgctcgt cgggcatcga
     7201 atctcgtcag ctatcgtcag cttacctttt tggcagcgat cgcggctccc gacatcttgg
     7261 accattagct ccacaggtat cttcttccct ctagtggtca taacagcagc ttcagctacc
     7321 tctcaattca aaaaacccct caagacccgt ttagaggccc caaggggtta tgctatcaat
     7381 cgttgcgtta cacacacaaa aaaccaacac acatccatct tcgatggata gcgattttat
     7441 tatctaactg ctgatcgagt gtagccagat ctagtaatca attacggggt cattagttca
     7501 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     7561 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     7621 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     7681 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     7741 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     7801 cgtattagtc atcgctatta ccatgctgat gcggttttgg cagtacatca atgggcgtgg
     7861 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
     7921 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
     7981 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa
     8041 ccgtcagatc agatctttgt cgatcctacc atccactcga cacacccgcc agcggccgc
//
